Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0056—Xeno-free medium
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/80—Undefined extracts from animals
  • C12N2500/84—Undefined extracts from animals from mammals

Definitions

• This invention relates to the culturing of mammalian cells.
• Mammalian cells are grown in culture for a variety of purposes.
• One increasingly important use of cultured mammalian cells is in the production of therapeutic proteins, e.g., tissue plasminogen activator, coded for by recombinant DNA - containing plasmids
• therapeutic proteins e.g., tissue plasminogen activator
• Examples of currently important production cell lines are mouse C127 cells and Chinese Hamster Ovary cells.
• mammaliam cells are cultured for therapeutic purposes.
• cancer treatment method described in Rosenberg et al. (1985) New Eng. J. Med. 313 , 1485.
• human lymphocytes are separated out of blood, cultured, treated with interleukin-2 to induce lymphocyte proliferation and activation, and infused into the patient so that the tumor is attacked by the activated lymphocytes.
• a superior, economical mammalian cell culture medium can be provided by using, rather than serum, a human plasma fraction, known as Cohn Supernatant Fraction V, which is inexpensive, stable under virus-killing heating conditions (60°C for 20 hours), and is very effective in promoting growth of non-adherent lymphoid and hybridoma cells and maintenance of adherent cells such as human fibroblasts.
• Cohn Supernatant Fraction V which is inexpensive, stable under virus-killing heating conditions (60°C for 20 hours)
• Supernatant Fraction V is a fraction produced during Method 6 of the Cohn Plasma Fractionation Process (described in Cohn et al. J. Am. Chem. Soc. 68 , 459 (1946), and later in Hormones in Human Plasma , Harry N. Antoniades, ed., Little Brown and Company, Boston, 19 ).
• the Cohn process is carried out by licensed laboratories according to the Bureau of Biological Standards's strictly defined procedures which, from time to time, with government approval, are varied slightly.
• Supernatant Fraction V the fraction which we have discovered is valuable in mammalian cell culture, is commonly discarded.
• Supernatant Fraction V is meant, herein, the fraction identified as such in the Cohn Fractionation Process, or the equivalent fraction produced as a result of the carrying out of an equivalent process on human plasma.
• the Cohn process as outlined in Antoniades, id, is reprinted below.
• the first step in preparing a cell growth medium of the invention is to isolate the protein-rich Supernatant Fraction V from human plasma using Cohn cold ethanol fractionation method 6.
• this involves a series of successive fractionation steps using aqueous ethanol fractionation solutions; the solutions vary in ethanol concentration, ionic strength, pH, and protein concentration.
• Each fractionation step which involves treatment with an appropriate fractionation solution followed by centrifugation, produces a plasma fraction having a distinctive distribution of plasma proteins.
• the ethanol concentration, ionic strength, pH, and protein concentration of each fractionation solution, as well as the temperature of each fractionation step, are chosen so as to maximize the stability of the fractionated proteins. Fractionation continues until Supernatant Fraction VI is isolated.
• Supernatant Fraction V Once Supernatant Fraction V has been isolated, it is filtered in one or more ultrafiltration steps to yield a protein fraction in which the proteins hace molecular weights greater than about 30 KD; the proteins consist primarily of albumin (about 80%). The protein fraction is then sterilized to remove bacteria and hepatitis and other viruses by heat treatment at about 60°C for approximately 10-20 hrs, followed by filtration through a sterile filter. The final protein concentration is about 4-10 g protein /100 ml of solution.
• the sterilized protein is then mixed with a conventional serum-free medium containing inorganic salts (electrolytes, etc.), amino acids, and vitamins required for cell growth, e.g., RPMI 1640, RPMI plus HEPES, Dulbecco's Modified Medium, etc., to provide the cell growth medium of the invention.
• a conventional serum-free medium containing inorganic salts (electrolytes, etc.), amino acids, and vitamins required for cell growth, e.g., RPMI 1640, RPMI plus HEPES, Dulbecco's Modified Medium, etc.
• the protein concentration in the complete medium is about 100-2000 ⁇ g/ml, preferably about 400 ⁇ g/ml.
• the resulting medium can be used to grow and maintain a variety of mammaliam cells, including the following general types of cells: 1) lymphoid cells; 2) anchorage-dependent cells; and 3) hybridoma cells. Conventional cell-culturing techniques and conditions are used.
• Human plasma (New York Blood Center) was treated at -3°C with a fractionation solution having the following composition: a) Ethanol - 8% b) Ionic Concentration ( ⁇ /2) - 0.14 c) pH - 7.2. The protein content of the resulting mixture was 5.1%. The mixture was then separated and the supernatant (Supernatant I) saved. Supernatant I was treated at -5° C with a fractionation solution having the following composition: a) Ethanol - 25% b) Ionic Concentration ( ⁇ /2) - 0.09 c) pH 6.9. The protein content of the resulting mixture was 3.0 %. The mixture was then separated and the supernatant (Supernatant II & III) saved.
• a fractionation solution having the following composition: a) Ethanol - 8% b) Ionic Concentration ( ⁇ /2) - 0.14 c) pH - 7.2. The protein content of the resulting mixture was 5.1%. The mixture was then separated and the supernatant
• the protein content of the resulting mixture was 1.6%.
• the mixture was then separated and the supernatant (Supernatant IV-1) saved.
• Supernatant IV-1 was treated at -5° C with a fractionation solution having the following composition: a) Ethanol - 40% b) Ionic Concentration ( ⁇ /2)-0.09 c) pH - 5.8. The protein content of the resulting solution was 1.0%. The mixture was then spearated and the supernatant (Supernatant IV-4) saved.
• Fraction V containing mostly albumin, was treated at -3° C with a fractionation solution having the following composition: a) Ethanol - 10% b) Ionic Concentration ( ⁇ /2) - 0.1 c) pH - 4.5.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=25439603

Family Applications (1)

Country Status (2)

Cited By (2)

Citations (1)

• 1987
  • 1987-10-09 JP JP62256121A patent/JPS63185376A/ja active Pending
  • 1987-10-10 EP EP19870114804 patent/EP0264748A3/de not_active Withdrawn

Patent Citations (1)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events